Diabetics are four to six times more likely to develop cataracts at a younger age than the normal population. Delayed or decreased incidence of such complications are seen as glucose levels are brought toward normal range. Glycation of vital proteins is one of the means by which elevated glucose can affect tissues such as lens that are insulin independent. Modification of specific amino acid residues of crystallins by glycation leads to disruption of protein organization. Increased exposure of sulfhydryl groups for eventual oxidation also may occur. This mechanism could play a significant role either in the initiation or in the enhancement of high molecular weight (HMW) aggregate formation, protein insolubilization, opacification, and cataract development. To establish the relationship between crystallin glycation and cataract development we propose to use streptozotocin diabetic rats to study the progressive changes in glycation, high molecular weight (HMW) aggregate formation, and protein insolubilization during precataract and cataract stages and to study the influence of aldose reductase inhibitors on lens protein glycation and HMW aggregate formation. In addition to animal studies, long-term in vitro glycation studies will be utilized to establish the relationship between glycation and HMW aggregation. The HMW proteins and free crystallins or other proteins of the water-soluble and urea- soluble fractions will be separated by molecular sieve high performance liquid chromatography (HPLC). Characterization of the proteins will involve utilization of reverse-phase HPLC for separation of protein subunits, amino acid analysis, isoelectric focusing, gel electrophoresis and immunoblotting. Thiol-disulfide exchange chromatography will be utilized for determining the extent of thiol oxidation or disulfide formation. The extent of glycation of the total water-soluble and urea-soluble fractions, the individual crystallins, and the aggregates will be determined by a combination of (3H)NaBH4 reduction, phenylboranate- agarose affinity chromatography and molecular sieve HPLC. The sites of glycation of each crystallin subunit will be identified. The presence of the ultimate products of glycation or the so- called fluorescent browning products will be monitored with a flourescence spectrophotometer. The proposed studies are expected to confirm the hypothesis: glycation-protein conformational change-increased reactivity of thiols-protein disulfides-aggregation.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Research Project (R01)
Project #
1R01EY007394-01
Application #
3264374
Study Section
Visual Sciences A Study Section (VISA)
Project Start
1987-09-30
Project End
1991-09-29
Budget Start
1987-09-30
Budget End
1988-09-29
Support Year
1
Fiscal Year
1987
Total Cost
Indirect Cost
Name
Medical College of Georgia (MCG)
Department
Type
Schools of Medicine
DUNS #
City
Augusta
State
GA
Country
United States
Zip Code
30912
Thampi, Prajitha; Zarina, Shamshad; Abraham, Edathara C (2002) alpha-Crystallin chaperone function in diabetic rat and human lenses. Mol Cell Biochem 229:113-8
Bera, Sibes; Abraham, Edathara C (2002) The alphaA-crystallin R116C mutant has a higher affinity for forming heteroaggregates with alphaB-crystallin. Biochemistry 41:297-305
Shroff, N P; Bera, S; Cherian-Shaw, M et al. (2001) Substituted hydrophobic and hydrophilic residues at methionine-68 influence the chaperone-like function of alphaB-crystallin. Mol Cell Biochem 220:127-33
Shroff, N P; Cherian-Shaw, M; Bera, S et al. (2000) Mutation of R116C results in highly oligomerized alpha A-crystallin with modified structure and defective chaperone-like function. Biochemistry 39:1420-6
Zarina, S; Zhao, H R; Abraham, E C (2000) Advanced glycation end products in human senile and diabetic cataractous lenses. Mol Cell Biochem 210:29-34
Swamy-Mruthinti, S; Shaw, S M; Zhao, H R et al. (1999) Evidence of a glycemic threshold for the development of cataracts in diabetic rats. Curr Eye Res 18:423-9
Cherian-Shaw, M; Smith, J B; Jiang, X Y et al. (1999) Intrapolypeptide disulfides in human alphaA-crystallin and their effect on chaperone-like function. Mol Cell Biochem 199:163-7
Zhao, H R; Nagaraj, R H; Abraham, E C (1997) The role of alpha- and epsilon-amino groups in the glycation-mediated cross-linking of gammaB-crystallin. Study of three site-directed mutants. J Biol Chem 272:14465-9
Matsumoto, K; Ikeda, K; Horiuchi, S et al. (1997) Immunochemical evidence for increased formation of advanced glycation end products and inhibition by aminoguanidine in diabetic rat lenses. Biochem Biophys Res Commun 241:352-4
Cherian, M; Smith, J B; Jiang, X Y et al. (1997) Influence of protein-glutathione mixed disulfide on the chaperone-like function of alpha-crystallin. J Biol Chem 272:29099-103

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